PROJECT SUMMARY Genome sequencing has catalogued the somatic alterations in human cancers and identified many putative driver genes. However, human cancers generally evolve through the sequential acquisition of multiple genomic alterations and simply identifying recurrent genomic alterations does not necessarily reveal their functional importance to cancer growth. Genetically engineered mouse models have become a mainstay for the analysis of gene function in cancer in vivo, however the breadth of their utility is limited by the fact that they are neither readily scalable nor sufficiently quantitative. To increase the scope and precision of in vivo cancer modeling, we previously integrated conventional genetically-engineered mouse models, CRISPR/Cas9-based somatic genome engineering, and quantitative genomics with mathematical approaches. We developed methods to inactivate multiple genes in parallel in mouse models of lung cancer using pools of barcoded sgRNA- containing lentiviral vectors. This tumor barcoding with sequencing (Tuba-seq) approach uncovers the size of each tumor, enables the parallel investigation of multiple tumor genotypes in individual mice, and allows the generation of large-scale maps of gene function within autochthonous cancer models. Our preliminary data and novel genetic systems, as well as our dedicated and collaborative team of investigators with expertise in cancer genetics, mouse models, genome-editing, clinical cancer care, and quantitative modeling make us uniquely positioned to conduct these studies. In this proposal, we will extend Tuba-seq to quantify the effect of combinatorial genetic alterations through the development and validation of a platform for the rapid and quantitative analysis of interactions between genetic alterations on tumor growth in vivo. To enable multiplexed and quantitative analysis of the impact of temporally controlled genomic alterations on cancer cell growth in vivo, we will also develop a system for inducible genome editing in established lung tumors. Finally, we will develop novel in vivo approaches to comprehensively and broadly uncover the gene expression programs in cancer cells of different genotypes in parallel. Through multiplexed in vivo genetic alterations, the effect of putative cancer drivers can be uncovered at an unprecedented scale and resolution. The results of this proposal will be significant because innovative methods for the cost-effective, quantitative, and multiplexed analysis of the genetic determinants of cancer pathogenesis will illuminate novel aspects of tumorigenesis and accelerate our ability to understand cancer evolution, drug responses, and therapy resistance.